Simian TRIM5α proteins produce replication of herpes simplex virus.
Herpes Simplex Virus (HSV) is a human nuclear DNA virus. This virus has a number of mechanisms to withstand blocks to replication within the host cell and so it can replicate in many different cell types as well. HSV is also capable of infecting many different species.
Tripartite motif (TRIM) is a protein family involved in various cellular processes. TRIM proteins affect different stages of viral life cycle by its actions in the nucleus and in the cytoplasm. TRIM19/PML is a component of ND10 bodies. TRIM19/PML interferes with replication of a number of DNA and RNA viruses including HSV-1. Experimental results suggest TRIM19/PML being an antiviral, part of the innate response to viral infections. One of the mechanisms HSV uses to overcome intracellular blocks to replication is disruption of this TRIM19/PML protein of ND10 bodies thus preventing restriction of its replication. (1)
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Although HSV has the ability to infect cells of many different animal species, infection of rhesus macaques does not lead to significant disease. The question addressed in this set of experiments was whether TRIM5α proteins have an effect on HSV replication. If so, it was further hypothesized that TRIM5α proteins could have an antiviral effect that could inhibit viruses outside of retrovirus family. (1)
Synopsis of Data
Based on several reports, rhesus macaques are less susceptible to HSV infection than other primates and are poor non-human primate models of HSV-1 and HSV-2 infection. To test validity of these reports, the ability of HSV-1 and HSV-2 to replicate in rhesus monkey fibroblasts were measured. The results were compared with the replication of those viruses in permissive HeLa cells. HeLa cells and rhesus macaque fibroblast cell lines were infected with HSV-1 and HSV-2 at an MOI of 3PFU/cell. Total virus was harvested at various times; the yields were determined by plaque assay on Vero cells. The results suggested that HSV-1 and HSV-2 yields obtained from rhesus monkey fibroblasts were significantly reduced (by about 100-fold) when compared to those from HeLa cells. These results led to further investigation whether the rhesus TRIM5α protein played a role in this replication decrease. (1)
HSV infection of HeLa cells that simultaneously expressed the rhesus monkey TRIM5α protein, (H-R cells) were examined in order to determine if TRIM5α protein played a role in the restriction of HSV-1 and HSV-2 rhesus cells. H-R cells that expressed rhesus monkey TRIM5α or the H-L control cells were infected with HSV-1 KOS strain or with HSV-2 186 syn+ strain at an MOI of 1, 3, 10 or 30 PFU/cell. The infections were harvested at 24hpi, and viral yields were measured by plaque assay on Vero cells. When the MOI levels were low, TRIM5α proteins were successful in reducing HSV replication. However, as MOI levels increased, TRIM5α proteins were no longer effective in the reduction of HSV replication. (1)
The effects of TRIM5α molecules from African green monkey, squirrel monkey, rhesus monkey and human were compared. HSV-2 yields were collected from cell lines that steadily express different TRIM5α variants. H-R (rhesus monkey), H-H (human), H-AGM (African green monkey), H-Sq (squirrel monkey) and H-L (control empty vector) cells were infected with HSV-2 186syn+ strain at an MOI of 3 PFU/cell and harvested after 24h. Viral yields were measured on Vero cells. The results showed that of all TRIM5α molecules experimented OWM TRIM5α molecules had the inhibitoriest effect on HSV replication. (1)
The restrictive activity of TRIM5α protein on retroviruses is thought to occur at an early stage of infection. Based on this it was hypothesized that rhesus monkey TRIM5α might apply its activity on HSV infection at an early stage. To test the hypothesis that HSV IE viral protein synthesis would be decreased in rhesus monkey TRIM5α-expressing cell lines, H-L control cells and H-R cells expressing rhesus monkey TRIM5α were infected with HSV-1 and HSV-2. Viral protein synthesis was assayed by Western blot. Based on the results, the immediate early gene expression inhibition was the cause of reduction in HSV-1 replication in H-R cells. Synthesis of viral protein in H-R cells was greater in HSV-2 infected cells compared to HSV-1 gene expression. (1)
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It was further investigated whether the replication of clinical isolates of HSV-1 and HSV-2 strains would be restricted by rhesus TRIM5α protein. H-R cells that steadily express rhesus monkey TRIM5α or the control H-L cells were infected with HSV-2 186 syn+, G, or SD90-3P strains at an MOI of 3. The infections were harvested at 24hpi, and viral yields were measured by plaque assay on Vero cells. H-R cells that express rhesus monkey TRIM5α or the control H-L cells were infected with HSV-1 strains F and 17syn+ at an MOI of 3. The infections were harvested at 24hpi, and the viral yields were measured by plaque assay on Vero cells. Based on the results, TRIM5α inhibits HSV-1 and HSV-2. This inhibition appeared to be more specific to the type of strain rather than what cell species HSV infected. (1)
It was hypothesized that TRIM5α acted in similar fashion to PML. PML, according to recent reports, functions by retaining the HSV infected cell protein 0 (ICP0) in the cytoplasm and reduces HSV-1 replication. This hypothesis was tested by examining the distribution of ICP0 in HSV-1 KOS virus-infected H-L, H-R, and H-H cells. The results indicated that rhesus TRIM5α function by increasing the amounts of ICP0 in the cytoplasm. Infection of H-R cells with HSV-1 strain 17 led to the similar changes in ICP0 distribution. These results have presented that TRIM5α block ICP0 from entering the nucleus by keeping in it in the cytoplasm of the cell; this way it inhibits HSV infection via prevention of its nuclear functions. (1)
In order to determine whether increased cytoplasmic ICP0 in H-R cells inhibition might be due to cytoplasmic retention of ICP0 at initial stages of the infection, it was tested whether an ICP0 mutant virus showed decreased replication in H-R cells relative to H-L cells. In order to test this, H-L and H-R cells were infected with the ICP0 null and rescued viruses; the viral yields were measured at 24hpi. Results of this experiment suggested that the level of TRIM5α inhibition in HSV-infected cells appeared to be independent of ICP0 being present. (1)
In order to determine whether HSV infection affected TRIM5α levels, Western blot analysis on lysates from mock- or HSV-infected H-R and H-L cells were performed. Both HSV-1 and HSV-2 infection resulted in decreased levels of TRIM5α. It was further tested whether this decrease correlated with the restrictions of HSV replication by infecting or mock-infecting cells that express different TRIM5α molecules (H-L, H-R, H-Sq, H-H, H-AGM) with HSV-2 virus. The infected cultures were harvested at 4, 6 or 16 hpi. Based on the results it was concluded that the amount of TRIM5α protein was not directly correlated with the level of HSV replication restriction. In other words, by adding more TRIM5α protein would not lead to an increase in HSV replication inhibition. (1)
According to the experimental results HSV lacks the ability to cause serious forms of disease in rhesus macaques. The experimental results have also suggested that both HSV-1 and HSV-2 grow poorly in rhesus monkey fibroblast cells. The effects of TRIM5α protein on the inhibition of HSV replication were tested. It was found that TRIM5α proteins significantly inhibit HSV-1 replication. TRIM5α proteins from different animals were further used to test their effect on HSV replication in HeLa cells. Experimental results suggested that Old World Monkey TRIM5α proteins had the strongest inhibiting effect on HSV followed by AGM TRIM5α, which are significant in inhibiting HSV as well as its protein expression. Squirrel monkey TRIM5α proteins exhibited a modest effect followed by human TRIM5α proteins which showed little to no effect. (1)
The experimental results also suggested some of the mechanisms which TRIM5α proteins might use in preventing HSV disease. TRIM5α proteins are found in the cytoplasm of the cell and reduce the expression of immediate early genes of the HSV. TRIM5α proteins might also be affecting events happening once the virus enters the cell. There is a possibility they interact with VP16 virion transactivator and/or HSV ICP0 by preventing its entrance into the nucleus. (1)
PML, component of ND10, affects chromatin structure. It is a cytoplasmic protein and one of its uses is to retain nuclear HSV molecules, ICP0 and VP16, in the cytoplasm. The experimental results showed a decrease in PML levels lead to an increase in viral gene expression. Increasing PML levels had no effect on viral gene expression. (1)
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In conclusion, the findings of the experiments described in this article were relevant. It was important to understand the viral vector interaction with rhesus cells because of the use rhesus macaques serve. Namely, rhesus macaques are used as SIV reservoirs which are similar to human HIV infection. Therefore, studying this interaction would allow researchers to better understand the virus as well help in developing a vaccine for human HIV.(1) There were findings which opened the ways in which further investigations of HSV inhibition could be taken. Perhaps replication factors found in rhesus macaque nuclei are not compatible with those required by HSV. Or it could be possible that rhesus macaque nuclear pores lack appropriate receptors to allow viral genome entry into the nucleus thus preventing expression of IE genes and blocking the process of replication.
1. Reszka, Natalia, Zhou, Changhong, Song, Byeongwoon, Sodroski, Joseph G., Knipe, David M. “Simian TRIM5α proteins produce replication of herpes simplex virus.” Virology 398(2010) 243-250.